Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family with attenuated expression of the rcsA gene

ABSTRACT

The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus  Escherichia  or  Pantoea , which has been modified to attenuate expression of the rcsA gene.

This application is a continuation under 35 U.S.C. §120 of PCT PatentApplication No. PCT/JP2007/061237, filed May 28, 2007, which claimspriority under 35 U.S.C. §119 to Russian Patent Application No.2006118967, filed on Jun. 1, 2006, and U.S. Provisional PatentApplication No. 60/868,102, filed Dec. 1, 2006, the entireties of whichare incorporated by reference. The Sequence Listing filed electronicallyherewith is also hereby incorporated by reference in its entirety (FileName: US-289 Seq List; File Size: 6 KB; Date Created: Nov. 26, 2008).

FIELD OF THE INVENTION

The present invention relates to the microbiological industry, andspecifically to a method for producing an L-amino acid using a bacteriumof the Enterobacteriaceae family which has been modified to attenuateexpression of the rcsA gene.

BRIEF DESCRIPTION OF THE RELATED ART

Conventionally, L-amino acids are industrially produced by fermentationmethods utilizing strains of microorganisms obtained from naturalsources, or mutants thereof. Typically, the microorganisms are modifiedto enhance production yields of L-amino acids.

Many techniques to enhance L-amino acid production yields have beenreported, including transformation of microorganisms with recombinantDNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques forenhancing production yields include increasing the activities of enzymesinvolved in amino acid biosynthesis and/or desensitizing the targetenzymes of the feedback inhibition by the resulting L-amino acid (see,for example, WO 95/16042 or U.S. Pat. Nos. 4,346,170; 5,661,012 and6,040,160).

Another way to enhance L-amino acid production yields is to attenuateexpression of a gene or several genes involved in degradation of thetarget L-amino acid, genes diverting the precursors of the targetL-amino acid from the L-amino acid biosynthetic pathway, genes involvedin the redistribution of carbon, nitrogen, and phosphate fluxes, andgenes coding for toxins etc.

The rcsA gene encodes the RcsA protein, which is an unstable positiveregulator required for the synthesis of colanic acid capsularpolysaccharide in Escherichia coli. Degradation of the RcsA protein invivo depends on a ATP-dependent Lon protease. The DNA and proteinsequences are highly homologous to a rcsA gene and a protein fromKlebsiella pneumoniae and other species. The carboxy-terminal region ofRcsA contains a potential helix-turn-helix DNA-binding motif whichresembles the sequence found at the carboxy-terminus of RcsB, which isanother positive regulator of capsule synthesis, and in several othertranscriptional regulators including members of the LuxR family. Thestability of wild-type RcsA in vivo is increased by the presence ofmultiple copies of RcsB, while RcsA is degraded more rapidly in rcsBmutant hosts than in wild-type hosts. These results suggest that RcsAand RcsB interact in vivo, and are consistent with genetic experimentsthat indicate an interaction between RcsA and RcsB (Stout, V. et. al.,J. Bacteriol. 1991 March; 173(5): 1738-1747)).

RcsA is shown to activate its own expression, as seen by the100-fold-increased expression of a rcsA::lacZ transcriptional fusion instrains with high levels of RcsA protein, either due to a mutation inlon or due to overexpression of RcsA from a multicopy plasmid.Expression of the rcsA::lacZ fusion is increased in the absence of RcsB.In addition, the effects of H—NS, a histone-like protein, and RcsB onthe expression of rcsA are independent of each other. A sequence motif,conserved between the E. coli cps promoter and the Erwinia amylovora amspromoter and previously shown to be the RcsA-RcsB binding site, wasidentified in the rcsA promoter region and shown to be required forhigh-level expression of rcsA. (Ebel, W. and Trempy, J. E., J.Bacteriol., 181(2): 577-84 (1999)).

The role of DnaJ, a heat shock protein, in the degradation of RcsA wasinvestigated. While the turnover of RcsA is slowed in dnaJ mutant hosts,the RcsA protein that accumulates is largely aggregated. Thesimultaneous stabilization and aggregation of RcsA are consistent with amodel in which DnaJ (and possibly DnaK and GrpE as well) acts tostimulate proteolysis indirectly by helping to maintain RcsA in asoluble conformation. The effect of DnaJ on RcsA solubility is notlimited to the protein overproduced from the plasmid. It was found thatthe chromosomally-encoded RcsA is mostly soluble in lon⁻ hosts butinsoluble in lon⁻ dnaJ⁻ hosts; furthermore, insolubility is associatedwith a failure of RcsA to function in dnaJ mutants (Jubete, Y., et. al.,J. Biol. Chem., 271(48), 30798-803 (1996)).

But currently, there have been no reports of attenuating expression ofthe rcsA gene for the purpose of producing L-amino acids.

SUMMARY OF THE INVENTION

Aspects of the present invention include enhancing the productivity ofL-amino acid-producing strains and providing a method for producing anL-amino acid using these strains.

The above aspects were achieved by finding that attenuating expressionof the rcsA gene can enhance production of L-amino acids, such asL-threonine, L-lysine, L-cysteine, L-methionine, L-leucine,L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline,L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

The present invention provides a bacterium of the Enterobacteriaceaefamily which has an increased ability to produce amino acids, such asL-threonine, L-lysine, L-cysteine, L-methionine, L-leucine,L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline,L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

It is an aspect of the present invention to provide an L-aminoacid-producing bacterium of the Enterobacteriaceae family, wherein thebacterium has been modified to attenuate expression of the rcsA gene.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the expression of the rcsA gene isattenuated by inactivation of the rcsA gene.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the bacterium belongs to the genusEscherichia.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the bacterium belongs to the genus Pantoea.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said L-amino acid is selected from the groupconsisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said aromatic L-amino acid is selected fromthe group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said non-aromatic L-amino acid is selectedfrom the group consisting of L-threonine, L-lysine, L-cysteine,L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine,L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine,L-glutamic acid, L-proline, and L-arginine.

It is a further aspect of the present invention to provide a method forproducing an L-amino acid comprising:

-   -   cultivating the bacterium as described above in a medium, and    -   collecting said L-amino acid from the medium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said L-amino acid is selected from the groupconsisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said aromatic L-amino acid is selected from thegroup consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said non-aromatic L-amino acid is selected fromthe group consisting of L-threonine, L-lysine, L-cysteine, L-methionine,L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine,L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid,L-proline, and L-arginine.

The present invention is described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative positions of primers P1 and P2 on plasmidpACYC184, which is used for amplification of the cat gene.

FIG. 2 shows the construction of the chromosomal DNA fragment comprisingthe inactivated rcsA-gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Bacterium of the Present Invention

The bacterium of the present invention is an L-amino acid-producingbacterium of the Enterobacteriaceae family, wherein the bacterium hasbeen modified to attenuate expression of the rcsA gene.

“L-amino acid-producing bacterium” means a bacterium which has anability to produce and excrete an L-amino acid into a medium, when thebacterium is cultured in the medium.

The term “L-amino acid-producing bacterium” also means a bacterium whichis able to produce and cause accumulation of an L-amino acid in aculture medium in an amount larger than a wild-type or parental strainof the bacterium, for example, E. coli, such as E. coli K-12, andpreferably means that the bacterium is able to cause accumulation in amedium of an amount not less than 0.5 g/L, more preferably not less than1.0 g/L, of the target L-amino acid. The term “L-amino acid” includesL-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine,L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, and L-valine.

The term “aromatic L-amino acid” includes L-phenylalanine, L-tyrosine,and L-tryptophan. The term “non-aromatic L-amino acid” includesL-threonine, L-lysine, L-cysteine, L-methionine, L-leucine,L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline,and L-arginine. L-threonine, L-lysine, L-cysteine, L-leucine,L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline,and L-arginine are particularly preferred.

The Enterobacteriaceae family includes bacteria belonging to the generaEscherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus,Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc.Specifically, those classified into the Enterobacteriaceae according tothe taxonomy used by the NCBI (National Center for BiotechnologyInformation) database(www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used.A bacterium belonging to the genus Escherichia or Pantoea is preferred.

The phrase “a bacterium belonging to the genus Escherichia” means thatthe bacterium is classified into the genus Escherichia according to theclassification known to a person skilled in the art of microbiology.Examples of a bacterium belonging to the genus Escherichia as used inthe present invention include, but are not limited to, Escherichia coli(E. coli). The bacterium belonging to the genus Escherichia is notparticularly limited, however for example, bacteria described byNeidhardt, F. C. et al. (Escherichia coli and Salmonella typhimurium,American Society for Microbiology, Washington D.C., 1208, Table 1) areencompassed.

The phrase “a bacterium belonging to the genus Pantoea” means that thebacterium is classified as the genus Pantoea according to theclassification known to a person skilled in the art of microbiology.Some species of Enterobacter agglomerans have been recentlyre-classified into Pantoea agglomerans, Pantoea ananatis, Pantoeastewartii or the like, based on the nucleotide sequence analysis of 16SrRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).

The phrase “bacterium has been modified to attenuate expression of thercsA gene” means that the bacterium has been modified in such a way thatthe modified bacterium contains reduced amounts of the RcsA protein ascompared with an unmodified bacterium, or the modified bacterium isunable to synthesize the RcsA protein.

The phrase “inactivation of the rcsA gene” means that the modified geneencodes a completely inactive protein. It is also possible that themodified DNA region is unable to naturally express the gene due to adeletion of a part of the gene or of the gene entirely, shifting of thereading frame of the gene, introduction of missense/nonsensemutation(s), or modification of an adjacent region of the gene,including sequences controlling gene expression, such as promoter(s),enhancer(s), attenuator(s), ribosome-binding site(s), etc.

The level of gene expression can be estimated by measuring the amount ofmRNA transcribed from the gene using various known methods includingNorthern blotting, quantitative RT-PCR, and the like. The amount of theprotein coded by the gene can be measured by known methods includingSDS-PAGE followed by immunoblotting assay (Western blotting analysis)and the like.

The rcsA gene (synonyms-ECK1949, cpsR, b1951) encodes the RcsA protein(synonyms-CpsR, B1951). The rcsA gene of E. coli (nucleotides inpositions 2,021,992 to 2,022,615 in the GenBank accession numberNC_(—)000913.2; gi:49175990; SEQ ID NO: 1) is located between the genefliR and the gene dsrB on the chromosome of E. coli strain K-12. Thenucleotide sequence of the rcsA gene and the amino acid sequence of RcsAencoded by the rcsA gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2,respectively.

Since there may be some differences in DNA sequences between the generaor strains of the Enterobacteriaceae family, the rcsA gene to beinactivated on the chromosome is not limited to the gene shown in SEQ IDNo:1, but may include genes homologous to SEQ ID No:1 which encode avariant protein of the RcsA protein. The phrase “variant protein” meansa protein which has changes in the sequence, whether they are deletions,insertions, additions, or substitutions of amino acids, but stillmaintains the activity of the product as the RcsA protein. The number ofchanges in the variant protein depends on the position in the threedimensional structure of the protein or the type of amino acid residues.It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 in SEQID NO: 2. These changes in the variants are conservative mutations thatpreserve the function of the protein. In other words, these changes inthe variants can occur in regions of the protein which are not criticalfor the three dimensional structure of the protein. This is because someamino acids have high homology to one another so the three dimensionalstructure is not affected by such a change. A conservative mutation is amutation wherein substitution takes place mutually among Phe, Trp, Tyr,if the substitution site is an aromatic amino acid; among Leu, Ile, Val,if the substitution site is a hydrophobic amino acid; between Gln, Asn,if it is a polar amino acid; among Lys, Arg, His, if it is a basic aminoacid; between Asp, Glu, if it is an acidic amino acid; and between Ser,Thr, if it is an amino acid having a hydroxyl group. Typicalconservative mutations are conservative substitutions. Specific examplesof substitutions that are considered to be conservative include:substitution of Ala with Ser or Thr; substitution of Arg with Gln, His,or Lys; substitution of Asn with Glu, Gln, Lys, His, or Asp;substitution of Asp with Asn, Glu, or Gln; substitution of Cys with Seror Ala; substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg;substitution of Glu with Gly, Asn, Gln, Lys, or Asp; substitution of Glywith Pro; substitution of His with Asn, Lys, Gln, Arg, or Tyr;substitution of Ile with Leu, Met, Val, or Phe; substitution of Leu withIle, Met, Val, or Phe; substitution of Lys with Asn, Glu, Gln, His, orArg; substitution of Met with Ile, Leu, Val, or Phe; substitution of Phewith Trp, Tyr, Met, Ile, or Leu; substitution of Ser with Thr or Ala;substitution of Thr with Ser or Ala; substitution of Trp with Phe orTyr; substitution of Tyr with His, Phe, or Trp; and substitution of Valwith Met, Ile, or Leu. Substitutions, deletions, insertions, additions,or inversions and the like of the amino acids described above includenaturally occurred mutations (mutant or variant) depending ondifferences in species, or individual differences of microorganisms thatretain the rcsA gene. Such a gene can be obtained by modifying thenucleotide sequence shown in SEQ ID NO: 1 using, for example,site-directed mutagenesis, so that the site-specific amino acid residuein the protein encoded includes substitutions, deletions, insertions, oradditions.

Moreover, the protein variant encoded by the rcsA gene may be one whichhas a homology of not less than 80%, preferably not less than 90%, andmost preferably not less than 95%, with respect to the entire amino acidsequence shown in SEQ ID NO. 2.

Homology between two amino acid sequences can be determined using thewell-known methods, for example, the computer program BLAST 2.0, whichcalculates three parameters: score, identity and similarity.

Moreover, the rcsA gene may be a variant which hybridizes understringent conditions with the nucleotide sequence shown in SEQ ID NO: 1,or a probe which can be prepared from the nucleotide sequence understringent conditions, provided that it encodes a functional RcsA proteinprior to inactivation. “Stringent conditions” include those under whicha specific hybrid, for example, a hybrid having homology of not lessthan 60%, preferably not less than 70%, more preferably not less than80%, still more preferably not less than 90%, and most preferably notless than 95%, is formed and a non-specific hybrid, for example, ahybrid having homology lower than the above, is not formed. For example,stringent conditions are exemplified by washing one time or more,preferably two or three times at a salt concentration of 1×SSC, 0.1%SDS, preferably 0.1×SSC, 0.1% SDS at 60° C. Duration of washing dependson the type of membrane used for blotting and, as a rule, should be whatis recommended by the manufacturer. For example, the recommendedduration of washing for the Hybond™ N+ nylon membrane (Amersham) understringent conditions is 15 minutes. Preferably, washing may be performed2 to 3 times. The length of the probe may be suitably selected,depending on the hybridization conditions, in this specific case, it maybe about 100 bp to 1 kbp.

Expression of the rcsA gene can be attenuated by introducing a mutationinto the gene on the chromosome so that intracellular activity of theprotein encoded by the gene is decreased as compared with an unmodifiedstrain. Such a mutation on the gene can be replacement of one base ormore to cause amino acid substitution in the protein encoded by the gene(missense mutation), introduction of a stop codon (nonsense mutation),deletion of one or two bases to cause a frame shift, insertion of adrug-resistance gene, or deletion of a part of the gene or the entiregene (Qiu, Z. and Goodman, M. F., J. Biol. Chem., 272, 8611-8617 (1997);Kwon, D. H. et al, J. Antimicrob. Chemother., 46, 793-796 (2000)).Expression of the rcsA gene can also be attenuated by modifying anexpression regulating sequence such as the promoter, the Shine-Dalgarno(SD) sequence, etc. (WO95/34672, Carrier, T. A. and Keasling, J. D.,Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods may be employed to introduce amutation by gene recombination. A mutant gene encoding a mutant proteinhaving a decreased activity is prepared, and a bacterium to be modifiedis transformed with a DNA fragment containing the mutant gene. Then thenative gene on the chromosome is replaced with the mutant gene byhomologous recombination, and the resulting strain is selected. Suchgene replacement using homologous recombination can be conducted by themethod employing a linear DNA, which is known as “Red-drivenintegration” (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci.USA, 97, 12, p 6640-6645 (2000), WO2005/010175), or by methods employinga plasmid containing a temperature-sensitive replication control region(Proc. Natl. Acad. Sci., USA, 97, 12, p 6640-6645 (2000), U.S. Pat. Nos.6,303,383 and 5,616,480). Furthermore, the introduction of asite-specific mutation by gene replacement using homologousrecombination as set forth above can also be performed by using aplasmid lacking the ability to replicate in the host.

Expression of the gene can also be attenuated by insertion of atransposon or an IS factor into the coding region of the gene (U.S. Pat.No. 5,175,107), or by conventional methods, such as mutagenesis with UVirradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine)treatment.

Inactivation of the gene can also be performed by conventional methods,such as by mutagenesis using UV irradiation or nitrosoguanidine(N-methyl-N′-nitro-N-nitrosoguanidine), site-directed mutagenesis, genedisruption using homologous recombination, or/and insertion-deletionmutagenesis (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12:5978-83 and Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci.USA, 2000, 97:12: 6640-45) also called “Red-driven integration”.

The presence of the RcsA protein can be detected using polyclonalantibodies by the method described in (Ebel, W. and Trempy, J. E., J.Bacteriol., 181(2): 577-84 (1999)).

The presence or absence of the rcsA gene on the chromosome of abacterium can be detected by well-known methods, including PCR, Southernblotting, and the like. In addition, the level of expression of a genecan be estimated by measuring the amount of mRNA transcribed from thegene using various well-known methods, including Northern blotting,quantitative RT-PCR, and the like. The amount or molecular weight of theprotein encoded by the gene can be measured by well-known methods,including SDS-PAGE followed by immunoblotting assay (Western blottinganalysis), and the like.

Methods for preparation of plasmid DNA, digestion and ligation of DNA,transformation, selection of an oligonucleotide as a primer, and thelike may be ordinary methods well-known to one skilled in the art. Thesemethods are described, for instance, in Sambrook, J., Fritsch, E. F.,and Maniatis, T., “Molecular Cloning: A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press (1989).

L-Amino Acid-Producing Bacteria

As a bacterium which is modified to attenuate expression of the rcsAgene, bacteria which are able to produce either an aromatic or anon-aromatic L-amino acids may be used.

The bacterium can be obtained by attenuating expression of the rcsA genein a bacterium which inherently has the ability to produce L-aminoacids. Alternatively, the bacterium can be obtained by imparting theability to produce L-amino acids to a bacterium already having theattenuated expression of the rcsA gene.

L-Threonine-Producing Bacteria

Examples of parent strains for deriving the L-threonine-producingbacteria include, but are not limited to, strains belonging to the genusEscherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Pat. Nos.5,175,107, 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Pat. No.5,631,157), E. coli NRRL-21593 (U.S. Pat. No. 5,939,307), E. coli FERMBP-3756 (U.S. Pat. No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520(U.S. Pat. No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika(in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911A), and the like.

The strain TDH-6 is deficient in the thrC gene, as well as beingsucrose-assimilative, and the ilvA gene has a leaky mutation. Thisstrain also has a mutation in the rhtA gene, which imparts resistance tohigh concentrations of threonine or homoserine. The strain B-3996contains the plasmid pVIC40 which was obtained by inserting a thrA*BCoperon which includes a mutant thrA gene into a RSF1010-derived vector.This mutant thrA gene encodes aspartokinase homoserine dehydrogenase Iwhich has substantially desensitized feedback inhibition by threonine.The strain B-3996 was deposited on Nov. 19, 1987 in the All-UnionScientific Center of Antibiotics (Russia, 117105 Moscow, NagatinskayaStreet, 3-A) under the accession number RIA 1867. The strain was alsodeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) onApr. 7, 1987 under the accession number VKPM B-3996.

E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strainfor deriving L-threonine-producing bacteria. The strain B-5318 isprototrophic with regard to isoleucine, and a temperature-sensitivelambda-phage C1 repressor and PR promoter replaces the regulatory regionof the threonine operon in plasmid pVIC40. The strain VKPM B-5318 wasdeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) on May 3, 1990 under accession number of VKPMB-5318.

Preferably, the bacterium is additionally modified to enhance expressionof one or more of the following genes:

-   -   the mutant thrA gene which codes for aspartokinase homoserine        dehydrogenase I resistant to feed back inhibition by threonine;    -   the thrB gene which codes for homoserine kinase;    -   the thrC gene which codes for threonine synthase;    -   the rhtA gene which codes for a putative transmembrane protein;    -   the asd gene which codes for aspartate-β-semialdehyde        dehydrogenase; and    -   the aspC gene which codes for aspartate aminotransferase        (aspartate transaminase);

The thrA gene which encodes aspartokinase homoserine dehydrogenase I ofEscherichia coli has been elucidated (nucleotide positions 337 to 2799,GenBank accession no. NC_(—)000913.2, gi: 49175990). The thrA gene islocated between the thrL and thrB genes on the chromosome of E. coliK-12. The thrB gene which encodes homoserine kinase of Escherichia colihas been elucidated (nucleotide positions 2801 to 3733, GenBankaccession no. NC_(—)000913.2, gi: 49175990). The thrB gene is locatedbetween the thrA and thrC genes on the chromosome of E. coli K-12. ThethrC gene which encodes threonine synthase of Escherichia coli has beenelucidated (nucleotide positions 3734 to 5020, GenBank accession no.NC_(—)000913.2, gi: 49175990). The thrC gene is located between the thrBgene and the yaaX open reading frame on the chromosome of E. coli K-12.All three genes function as a single threonine operon. To enhanceexpression of the threonine operon, the attenuator region which affectsthe transcription is removed from the operon (WO2005/049808,WO2003/097839).

A mutant thrA gene which codes for aspartokinase homoserinedehydrogenase I resistant to feed back inhibition by threonine, as wellas the thrB and thrC genes can be obtained as one operon from thewell-known plasmid pVIC40 which is present in the threonine producing E.coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S.Pat. No. 5,705,371.

The rhtA gene exists at 18 min on the E. coli chromosome close to theglnHPQ operon, which encodes components of the glutamine transportsystem. The rhtA gene is identical to ORFI (ybiF gene, nucleotidepositions 764 to 1651, GenBank accession number AAA218541, gi:440181)and is located between the pexB and ompX genes. The unit expressing aprotein encoded by the ORFI has been designated the rhtA gene (rht:resistance to homoserine and threonine). Also, it was revealed that therhtA23 mutation is an A-for-G substitution at position-1 with respect tothe ATG start codon (ABSTRACTS of the 17^(th) International Congress ofBiochemistry and Molecular Biology in conjugation with the AnnualMeeting of the American Society for Biochemistry and Molecular Biology,San Francisco, Calif. Aug. 24-29, 1997, abstract No. 457, EP 1013765 A).

The asd gene of E. coli has already been elucidated (nucleotidepositions 3572511 to 3571408, GenBank accession no. NC_(—)000913.1,gi:16131307), and can be obtained by PCR (polymerase chain reaction;refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizingprimers prepared based on the nucleotide sequence of the gene. The asdgenes of other microorganisms can be obtained in a similar manner.

Also, the aspC gene of E. coli has already been elucidated (nucleotidepositions 983742 to 984932, GenBank accession no. NC_(—)000913.1,gi:16128895), and can be obtained by PCR. The aspC genes of othermicroorganisms can be obtained in a similar manner.

L-Lysine-Producing Bacteria

Examples of L-lysine-producing bacteria belonging to the genusEscherichia include mutants having resistance to an L-lysine analogue.The L-lysine analogue inhibits growth of bacteria belonging to the genusEscherichia, but this inhibition is fully or partially desensitized whenL-lysine coexists in a medium. Examples of the L-lysine analogueinclude, but are not limited to, oxalysine, lysine hydroxamate,S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam,and so forth. Mutants having resistance to these lysine analogues can beobtained by subjecting bacteria belonging to the genus Escherichia to aconventional artificial mutagenesis treatment. Specific examples ofbacterial strains useful for producing L-lysine include Escherichia coliAJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) andEscherichia coli VL611. In these microorganisms, feedback inhibition ofaspartokinase by L-lysine is desensitized.

The strain WC196 may be used as an L-lysine producing bacterium ofEscherichia coli. This bacterial strain was bred by conferring AECresistance to the strain W3110, which was derived from Escherichia coliK-12. The resulting strain was designated Escherichia coli AJ13069 andwas deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (currentlyNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary, Tsukuba Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Dec. 6,1994 and received an accession number of FERM P-14690. Then, it wasconverted to an international deposit under the provisions of theBudapest Treaty on Sep. 29, 1995, and received an accession number ofFERM BP-5252 (U.S. Pat. No. 5,827,698).

Examples of parent strains for deriving L-lysine-producing bacteria alsoinclude strains in which expression of one or more genes encoding anL-lysine biosynthetic enzyme are enhanced. Examples of such genesinclude, but are not limited to, genes encoding dihydrodipicolinatesynthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase(dapB), diaminopimelate decarboxylase (lysA), diaminopimelatedehydrogenase (ddh) (U.S. Pat. No. 6,040,160), phosphoenolpyruvatecarboxylase (ppc), aspartate semialdehyde dehydrogenease (asd), andaspartase (aspA) (EP 1253195 A). In addition, the parent strains mayhave increased expression of the gene involved in energy efficiency(cyo) (EP 1170376 A), the gene encoding nicotinamide nucleotidetranshydrogenase (pntAB) (U.S. Pat. No. 5,830,716), the ybjE gene(WO2005/073390), or combinations thereof.

Examples of parent strains for deriving L-lysine-producing bacteria alsoinclude strains having decreased or eliminated activity of an enzymethat catalyzes a reaction for generating a compound other than L-lysineby branching off from the biosynthetic pathway of L-lysine. Examples ofthe enzymes that catalyze a reaction for generating a compound otherthan L-lysine by branching off from the biosynthetic pathway of L-lysineinclude homoserine dehydrogenase, lysine decarboxylase (U.S. Pat. No.5,827,698), and the malic enzyme

L-Cysteine-Producing Bacteria

Examples of parent strains for deriving L-cysteine-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia, such as E. coli JM15 which is transformed with differentcysE alleles coding for feedback-resistant serine acetyltransferases(U.S. Pat. No. 6,218,168, Russian patent application 2003121601); E.coli W3110 having over-expressed genes which encode proteins suitablefor secreting substances toxic for cells (U.S. Pat. No. 5,972,663); E.coli strains having lowered cysteine desulfohydrase activity(JP11155571A2); E. coli W3110 with increased activity of a positivetranscriptional regulator for cysteine regulon encoded by the cysB gene(WO0127307A1), and the like.

L-Leucine-Producing Bacteria

Examples of parent strains for deriving L-leucine-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia, such as E. coli strains resistant to leucine (for example,the strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121)) or leucine analogsincluding β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine,5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strainsobtained by the gene engineering method described in WO96/06926; E. coliH-9068 (JP 8-70879 A), and the like.

The bacterium may be improved by enhancing the expression of one or moregenes involved in L-leucine biosynthesis. Examples include genes of theleuABCD operon, which are preferably represented by a mutant leuA genecoding for isopropylmalate synthase which is freed from feedbackinhibition by L-leucine (U.S. Pat. No. 6,403,342). In addition, thebacterium may be improved by enhancing the expression of one or moregenes coding for proteins which excrete L-amino acid from the bacterialcell. Examples of such genes include the b2682 and b2683 genes (ygaZHgenes) (EP 1239041 A2).

L-Histidine-Producing Bacteria

Examples of parent strains for deriving L-histidine-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. colistrain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116-B12121 (U.S.Pat. No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERMBP-6676) (U.S. Pat. No. 6,344,347); E. coli H-9341 (FERM BP-6674)(EP1085087); E. coli A180/pFM201 (U.S. Pat. No. 6,258,554) and the like.

Examples of parent strains for deriving L-histidine-producing bacteriaalso include strains in which expression of one or more genes encodingan L-histidine biosynthetic enzyme are enhanced. Examples of such genesinclude genes encoding ATP phosphoribosyltransferase (hisG),phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATPpyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazolecarboxamide ribotide isomerase (hisA), amidotransferase (hisH),histidinol phosphate aminotransferase (hisC), histidinol phosphatase(hisB), histidinol dehydrogenase (hisD), and so forth.

It is known that the L-histidine biosynthetic enzymes encoded by hisGand hisBHAFI are inhibited by L-histidine, and therefore anL-histidine-producing ability can also be efficiently enhanced byintroducing a mutation into ATP phosphoribosyltransferase which impartsresistance to the feedback inhibition (Russian Patent Nos. 2003677 and2119536).

Specific examples of strains having an L-histidine-producing abilityinclude E. coli FERM P-5038 and 5048 which have been introduced with avector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP56-005099 A), E. coli strains introduced with rht, a gene for an aminoacid-export (EP1016710A), E. coli 80 strain imparted withsulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance(VKPM B-7270, Russian Patent No. 2119536), and so forth.

L-Glutamic Acid-Producing Bacteria

Examples of parent strains for deriving L-glutamic acid-producingbacteria include, but are not limited to, strains belonging to the genusEscherichia, such as E. coli VL334thrC⁺ (EP 1172433). E. coli VL334(VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strainhaving mutations in thrC and ilvA genes (U.S. Pat. No. 4,278,765). Awild-type allele of the thrC gene was transferred by the method ofgeneral transduction using a bacteriophage P1 which was grown onwild-type E. coli K12 (VKPM B-7) cells. As a result, an L-isoleucineauxotrophic strain VL334thrC⁺ (VKPM B-8961), which is able to produceL-glutamic acid, was obtained.

Examples of parent strains for deriving the L-glutamic acid-producingbacteria include, but are not limited to, strains which are deficient inα-ketoglutarate dehydrogenase activity, or strains in which one or moregenes encoding an L-glutamic acid biosynthetic enzyme are enhanced.Examples of the genes involved in L-glutamic acid biosynthesis includegenes encoding glutamate dehydrogenase (gdhA), glutamine synthetase(glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA),aconitate hydratase (acnA, acnB), citrate synthase (gltA),phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc),pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pyka, pykF),phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase(pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphatedehydrogenase (gapA), triose phosphate isomerase (tpiA), fructosebisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), andglucose phosphate isomerase (pgi).

Examples of strains modified so that expression of the citratesynthetase gene, the phosphoenolpyruvate carboxylase gene, and/or theglutamate dehydrogenase gene is/are enhanced include those disclosed inEP1078989A, EP955368A, and EP952221A.

Examples of strains which have been modified so that expression of thecitrate synthetase gene and/or the phosphoenolpyruvate carboxylase geneare reduced, and/or/are deficient in α-ketoglutarate dehydrogenaseactivity include those disclosed in EP1078989A, EP955368A, andEP952221A.

Examples of parent strains for deriving the L-glutamic acid-producingbacteria also include strains having decreased or eliminated activity ofan enzyme that catalyzes synthesis of a compound other than L-glutamicacid by branching off from an L-glutamic acid biosynthesis pathway.Examples of such enzymes include isocitrate lyase (aceA),α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta),acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactatesynthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase(ldh), and glutamate decarboxylase (gadAB). Bacteria belonging to thegenus Escherichia deficient in α-ketoglutarate dehydrogenase activity orhaving a reduced α-ketoglutarate dehydrogenase activity and methods forobtaining them are described in U.S. Pat. Nos. 5,378,616 and 5,573,945.Specifically, these strains include the following:

E. coli W311 OsucA:: Km^(R)

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA::Km^(R) is a strain obtained by disrupting theα-ketoglutarate dehydrogenase gene (hereinafter referred to as “sucAgene”) of E. coli W3110. This strain is completely deficient inα-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacteria include those whichbelong to the genus Escherichia and have resistance to an aspartic acidantimetabolite. These strains can also be deficient in α-ketoglutaratedehydrogenase activity and include, for example, E. coli AJ13199 (FERMBP-5807) (U.S. Pat. No. 5,908,768), FFRM P-12379, which additionally hasa low L-glutamic acid decomposing ability (U.S. Pat. No. 5,393,671);AJ13138 (FERM BP-5565) (U.S. Pat. No. 6,110,714), and the like.

Examples of L-glutamic acid-producing bacteria, include mutant strainsbelonging to the genus Pantoea which are deficient in α-ketoglutaratedehydrogenase activity or have decreased α-ketoglutarate dehydrogenaseactivity, and can be obtained as described above. Such strains includePantoea ananatis AJ13356. (U.S. Pat. No. 6,331,419). Pantoea ananatisAJ13356 was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry (currently, National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, 305-8566, Japan) on Feb. 19, 1998 under an accession numberof FERM P-16645. It was then converted to an international deposit underthe provisions of Budapest Treaty on Jan. 11, 1999 and received anaccession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficientin α-ketoglutarate dehydrogenase activity as a result of disruption ofthe αKGDH-E1 subunit gene (sucA). The above strain was identified asEnterobacter agglomerans when it was isolated and deposited asEnterobacter agglomerans AJ13356. However, it was recently re-classifiedas Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNAand so forth. Although AJ13356 was deposited at the aforementioneddepository as Enterobacter agglomerans, for the purposes of thisspecification, they are described as Pantoea ananatis.

L-Phenylalanine-Producing Bacteria

Examples of parent strains for deriving L-phenylalanine-producingbacteria include, but are not limited to, strains belonging to the genusEscherichia, such as E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197);E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Pat.No. 5,354,672); E. coli MWEC101-b (KR8903681); E. coli NRRL B-12141,NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Pat. No. 4,407,952).Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERMBP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coliK-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110(tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used(EP 488424 B1). Furthermore, L-phenylalanine producing bacteriabelonging to the genus Escherichia with an enhanced activity of theprotein encoded by the yedA gene or the yddG gene may also be used (U.S.patent applications 2003/0148473 A1 and 2003/0157667 A1).

L-Tryptophan-Producing Bacteria

Examples of parent strains for deriving the L-tryptophan-producingbacteria include, but are not limited to, strains belonging to the genusEscherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91(DSM10123) which is deficient in the tryptophanyl-tRNA synthetaseencoded by mutant trpS gene (U.S. Pat. No. 5,756,345); E. coli SV164(pGH5) having a serA allele encoding phosphoglycerate dehydrogenase freefrom feedback inhibition by serine and a trpE allele encodinganthranilate synthase free from feedback inhibition by tryptophan (U.S.Pat. No. 6,180,373); E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50) aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S.Pat. No. 4,371,614); E. coli AGX17/pGX50, pACKG4-pps in which aphosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Pat.No. 6,319,696), and the like may be used. L-tryptophan-producingbacteria belonging to the genus Escherichia with an enhanced activity ofthe identified protein encoded by and the yedA gene or the yddG gene mayalso be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667A1).

Examples of parent strains for deriving the L-tryptophan-producingbacteria also include strains in which one or more activities of theenzymes selected from anthranilate synthase, phosphoglyceratedehydrogenase, and tryptophan synthase are enhanced. The anthranilatesynthase and phosphoglycerate dehydrogenase are both subject to feedbackinhibition by L-tryptophan and L-serine, so that a mutationdesensitizing the feedback inhibition may be introduced into theseenzymes. Specific examples of strains having such a mutation include aE. coli SV164 which harbors desensitized anthranilate synthase and atransformant strain obtained by introducing into the E. coli SV164 theplasmid pGH5 (WO 94/08031), which contains a mutant serA gene encodingfeedback-desensitized phosphoglycerate dehydrogenase.

Examples of parent strains for deriving the L-tryptophan-producingbacteria also include strains into which the tryptophan operon whichcontains a gene encoding desensitized anthranilate synthase has beenintroduced (JP 57-71397 A, JP 62-244382 A, U.S. Pat. No. 4,371,614).Moreover, L-tryptophan-producing ability may be imparted by enhancingexpression of a gene which encodes tryptophan synthase. The tryptophansynthase consists of α and β subunits which are encoded by the trpA andtrpB genes, respectively. In addition, L-tryptophan-producing abilitymay be improved by enhancing expression of the isocitrate lyase-malatesynthase operon (WO2005/103275).

L-Proline-Producing Bacteria

Examples of parent strains for deriving L-proline-producing bacteriainclude, but are not limited to, strains belonging to the genusEscherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient inthe ilvA gene and is able to produce L-proline (EP 1172433). Thebacterium may be improved by enhancing the expression of one or moregenes involved in L-proline biosynthesis. Examples of such genes forL-proline producing bacteria include the proB gene coding for glutamatekinase which has feedback inhibition by L-proline desensitized (DEPatent 3127361). In addition, the bacterium may be improved by enhancingthe expression of one or more genes coding for proteins excretingL-amino acid from bacterial cell. Such genes are exemplified by theb2682 and b2683 genes (ygaZH genes) (EP1239041 A2).

Examples of bacteria belonging to the genus Escherichia, which have anactivity to produce L-proline include the following E. coli strains:NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russianpatent application 2000124295), plasmid mutants described in DE Patent3127361, plasmid mutants described by Bloom F. R. et al (The 15^(th)Miami winter symposium, 1983, p. 34), and the like.

L-Arginine-Producing Bacteria

Examples of parent strains for deriving L-arginine-producing bacteria ofthe present invention include, but are not limited to, strains belongingto the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) (U.S.Patent Application 2002/058315 A1) and its derivative strains harboringmutant N-acetylglutamate synthase (Russian Patent Application No.2001112869), E. coli strain 382 (VKPM B-7926) (EP1170358A1), anarginine-producing strain into which argA gene encodingN-acetylglutamate synthetase is introduced therein (EP1170361A1), andthe like.

Examples of parent strains for deriving L-arginine producing bacteriaalso include strains in which expression of one or more genes encodingan L-arginine biosynthetic enzyme are enhanced. Examples of such genesinclude genes encoding N-acetylglutamyl phosphate reductase (argC),ornithine acetyl transferase (argJ), N-acetylglutamate kinase (argB),acetylornithine transaminase (argD), ornithine carbamoyl transferase(argF), argininosuccinic acid synthetase (argG), argininosuccinic acidlyase (argH), and carbamoyl phosphate synthetase (carAB).

L-Valine-Producing Bacteria

Example of parent strains for deriving L-valine-producing bacteriainclude, but are not limited to, strains which have been modified tooverexpress the ilvGMEDA operon (U.S. Pat. No. 5,998,178). It isdesirable to remove the region of the ilvGMEDA operon which is requiredfor attenuation so that expression of the operon is not attenuated bythe L-valine that is produced. Furthermore, the ilvA gene in the operonis desirably disrupted so that threonine deaminase activity isdecreased.

Examples of parent strains for deriving L-valine-producing bacteriainclude also include mutants having a mutation of amino-acyl t-RNAsynthetase (U.S. Pat. No. 5,658,766). For example, E. coli VL1970, whichhas a mutation in the ileS gene encoding isoleucine tRNA synthetase, canbe used. E. coli VL1970 has been deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1Dorozhny Proezd, 1) on Jun. 24, 1988 under accession number VKPM B-4411.

Furthermore, mutants requiring lipoic acid for growth and/or lackingH⁺-ATPase can also be used as parent strains (WO96/06926).

L-Isoleucine-Producing Bacteria

Examples of parent strains for deriving L-isoleucine producing bacteriainclude, but are not limited to, mutants having resistance to6-dimethylaminopurine (JP 5-304969 A), mutants having resistance to anisoleucine analogue such as thiaisoleucine and isoleucine hydroxamate,and mutants additionally having resistance to DL-ethionine and/orarginine hydroxamate (JP 5-130882 A). In addition, recombinant strainstransformed with genes encoding proteins involved in L-isoleucinebiosynthesis, such as threonine deaminase and acetohydroxate synthase,can also be used as parent strains (JP 2-458 A, FR 0356739, and U.S.Pat. No. 5,998,178).

2. Method of the Present Invention

The method of the present invention is a method for producing an L-aminoacid by cultivating the bacterium in a culture medium to produce andexcrete the L-amino acid into the medium, and collecting the L-aminoacid from the medium.

In the present invention, the cultivation, collection, and purificationof an L-amino acid from the medium and the like may be performed in amanner similar to conventional fermentation methods wherein an aminoacid is produced using a bacterium.

The chosen culture medium may be either a synthetic or natural medium,so long as it includes a carbon source and a nitrogen source andminerals and, if necessary, appropriate amounts of nutrients which thebacterium requires for growth. The carbon source may include variouscarbohydrates such as glucose and sucrose, and various organic acids.Depending on the mode of assimilation of the chosen microorganism,alcohol, including ethanol and glycerol, may be used. As the nitrogensource, various ammonium salts such as ammonia and ammonium sulfate,other nitrogen compounds such as amines, a natural nitrogen source suchas peptone, soybean-hydrolysate, and digested fermentativemicroorganisms can be used. As minerals, potassium monophosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,calcium chloride, and the like can be used. As vitamins, thiamine, yeastextract, and the like, can be used.

The cultivation is preferably performed under aerobic conditions, suchas by shaking and/or stirring with aeration, at a temperature of 20 to40° C., preferably 30 to 38° C. The pH of the culture is usually between5 and 9, preferably between 6.5 and 7.2. The pH of the culture can beadjusted with ammonia, calcium carbonate, various acids, various bases,and buffers. Usually, a 1 to 5-day cultivation leads to accumulation ofthe target L-amino acid in the liquid medium.

After cultivation, solids such as cells can be removed from the liquidmedium by centrifugation or membrane filtration, and then the L-aminoacid can be collected and purified by ion-exchange, concentration,and/or crystallization methods.

EXAMPLES

The present invention will be more concretely explained below withreference to the following non-limiting Examples.

Example 1 Construction of a Strain with an Inactivated rcsA Gene

1. Deletion of the rcsA Gene.

The rcsA gene was deleted by the method initially developed by Datsenko,K. A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12),6640-6645) called “Red-driven integration”. According to this procedure,the PCR primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4), which arecomplementary to both the region adjacent to the rcsA gene and the geneconferring antibiotic resistance, respectively, in the template plasmid,were constructed. The plasmid pACYC184 (NBL Gene Sciences Ltd., UK)(GenBank/EMBL accession number X06403) was used as a template in the PCRreaction. Conditions for PCR were as follows: denaturation step:3 min at95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C.,40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.

An 1152 bp PCR product (FIG. 1) was obtained and purified in agarose geland used for electroporation of E. coli MG1655 (ATCC 700926), whichcontains the plasmid pKD46 having a temperature-sensitive replication.The plasmid pKD46 (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad.Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotide (31088-33241)DNA fragment of phage λ (GenBank accession No. J02459), and containsgenes of the λ Red homologous recombination system (γ, β, exo genes)under the control of the arabinose-inducible P_(araB) promoter. Theplasmid pKD46 is necessary for integration of the PCR product into thechromosome of strain MG1655. The strain MG1655 can be obtained fromAmerican Type Culture Collection. (P.O. Box 1549 Manassas, Va. 20108,U.S.A.).

Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46was grown overnight at 30° C. in LB medium containing ampicillin (100mg/l), and the culture was diluted 100 times with 5 ml of SOB medium(Sambrook et al, “Molecular Cloning A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press (1989)) containingampicillin and L-arabinose (1 mM). The cells were grown with aeration at30° C. to an OD₆₀₀ of ≈0.6 and then were made electrocompetent byconcentrating 100-fold and washing three times with ice-cold deionizedH₂O. Electroporation was performed using 70 μl of cells and ≈100 ng ofPCR product. Cells after electroporation were incubated with 1 ml of SOCmedium (Sambrook et al, “Molecular Cloning A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press (1989)) at 37° C. for 2.5hours and were then plated onto L-agar containing chloramphenicol (30μg/ml) and grown at 37° C. to select Cm^(R) recombinants. Then, toeliminate the pKD46 plasmid, 2 passages on L-agar with Cm at 42° C. wereperformed and the obtained colonies were tested for sensitivity toampicillin.

2. Verification of the rcsA Gene Deletion by PCR.

The mutants, which have the rcsA gene deleted and are marked with the Cmresistance gene, were verified by PCR. Locus-specific primers P3 (SEQ IDNO: 5) and P4 (SEQ ID NO: 6) were used in PCR for verification.Conditions for PCR verification were as follows: denaturation step: 3min at 94° C.; profile for the 30 cycles: 30 sec at 94° C., 30 sec at54° C., 1 min at 72° C.; final step: 7 min at 72° C. The PCR productobtained using the parental rcsA⁺ strain MG1655 as the template, is 722bp in length. The PCR product obtained using the mutant strain as thetemplate is 1214 nucleotides in length (FIG. 2). The mutant strain wasnamed MG1655 Δ rcsA::cat.

Example 2 Production of L-threonine by E. coli strain B-3996-ΔrcsA

To test the effect of inactivation of the rcsA gene on threonineproduction, DNA fragments from the chromosome of the above-described E.coli MG1655 ΔrcsA::cat were transferred to the threonine-producing E.coli strain VKPM B-3996 by P1 transduction (Miller, J. H. Experiments inMolecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview,N.Y.) to obtain the strain B-3996-ΔrcsA. The strain B-3996 was depositedon Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics(Russia, 117105 Moscow, Nagatinskaya Street, 3-A) under the accessionnumber RIA 1867. The strain was also deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1Dorozhny proezd, 1) under the accession number VKPM B-3996.

Both E. coli B-3996 and B-3996-ΔrcsA, were grown for 18-24 hours at 37°C. on L-agar plates. To obtain a seed culture, the strains were grown ona rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubescontaining 2 ml of L-broth supplemented with 4% glucose. Then, thefermentation medium was inoculated with 0.21 ml (10%) of seed material.The fermentation was performed in 2 ml of minimal medium forfermentation in 20×200-mm test tubes. Cells were grown for 65 hours at32° C. with shaking at 250 rpm.

After cultivation, the amount of L-threonine, which had accumulated inthe medium, was determined by paper chromatography using the followingmobile phase: butanol:acetic acid:water=4:1:1 (v/v). A solution 2% ofninhydrin in acetone was used as a visualizing reagent. A spotcontaining L-threonine was cut out, L-threonine was eluted in 0.5% watersolution of CdCl₂, and the amount of L-threonine was estimatedspectrophotometrically at 540 nm. The results of 8 independent test tubefermentations are shown in Table 1. As follows from Table 1,B-3996-ΔrcsA caused accumulation of a higher amount of L-threonine, ascompared with B-3996.

The composition of the fermentation medium (g/l) was as follows:

Glucose 80.0 (NH₄)₂SO₄ 22.0 NaCl 0.8 KH₂PO₄ 2.0 MgSO₄•7H₂O 0.8FeSO₄•7H₂O 0.02 MnSO₄•5H₂O 0.02 Thiamine HCl 0.0002 Yeast extract 1.0CaCO₃ 30.0

Glucose and magnesium sulfate were sterilized separately. CaCO₃ wassterilized by dry-heat at 180° C. for 2 hours. The pH was adjusted to7.0. The antibiotic was introduced into the medium after sterilization.

TABLE 1 Strain OD₅₄₀ Amount of L-threonine, g/l B-3996 29.4 ± 0.7 25.9 ±0.7 B-3996-ΔrcsA 29.8 ± 0.5 27.7 ± 0.8

Example 3 Production of L-lysine by E. coli AJ11442-ΔrcsA

To test the effect of inactivation of the rcsA gene on lysineproduction, DNA fragments from the chromosome of the above-described E.coli strain MG1655 ΔrcsA::cat can be transferred to the lysine-producingE. coli strain AJ11442 by P1 transduction (Miller, J. H. Experiments inMolecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview,N.Y.) to obtain AJ11442-ΔrcsA strain. The strain AJ14442 was depositedat the National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology (currently National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1, 1981 and receivedan accession number of FERM P-5084. Then, it was converted to aninternational deposit under the provisions of the Budapest Treaty onOct. 29, 1987, and received an accession number of FERM BP-1543.

Both E. coli strains, AJ11442 and AJ11442-ΔrcsA, can be cultured inL-medium containing streptomycin (20 mg/l) at 37° C., and 0.3 ml of theobtained culture can be inoculated into 20 ml of the fermentation mediumcontaining the required drugs in a 500-ml flask. The cultivation can becarried out at 37° C. for 16 h by using a reciprocal shaker at theagitation speed of 115 rpm. After the cultivation, the amounts ofL-lysine and residual glucose in the medium can be measured by a knownmethod (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then,the yield of L-lysine can be calculated relative to consumed glucose foreach of the strains.

The composition of the fermentation medium (g/l) is as follows:

Glucose 40 (NH₄)₂SO₄ 24 K₂HPO₄ 1.0 MgSO₄•7H₂O 1.0 FeSO₄•7H₂O 0.01MnSO₄•5H₂O 0.01 Yeast extract 2.0

The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115° C.for 10 min. Glucose and MgSO₄.7H₂O are sterilized separately. CaCO₃ isdry-heat sterilized at 180° C. for 2 hours and added to the medium for afinal concentration of 30 μl.

Example 4 Production of L-cysteine by E. coli JM15 (ydeD)-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-cysteineproduction, DNA fragments from the chromosome of the above-described E.coli MG1655 ΔrcsA::cat can be transferred to the E. coliL-cysteine-producing strain JM15 (ydeD) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,1972, Plainview, N.Y.) to obtain the strain JM15 (ydeD)-ΔrcsA.

E. coli JM15 (ydeD) is a derivative of E. coli JM15 (U.S. Pat. No.6,218,168), which can be transformed with DNA having the ydeD geneencoding a membrane protein, and is not involved in a biosyntheticpathway of any L-amino acid (U.S. Pat. No. 5,972,663). The strain JM15(CGSC# 5042) can be obtained from The Coli Genetic Stock Collection atthe E. coli Genetic Resource Center, MCD Biology Department, YaleUniversity (cgsc.biology.yale.edu/).

Fermentation conditions for evaluation of L-cysteine production weredescribed in detail in Example 6 of U.S. Pat. No. 6,218,168.

Example 5 Production of L-leucine by E. coli 57-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-leucineproduction, DNA fragments from the chromosome of the above-described E.coli strain MG1655 ΔrcsA::cat can be transferred to the E. coliL-leucine-producing strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121) byP1 transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain57-pMW-ΔrcsA. The strain 57 has been deposited in the Russian NationalCollection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1Dorozhny proezd, 1) on May 19, 1997 under accession number VKPM B-7386.

Both E. coli strains, 57 and 57-ΔrcsA, can be cultured for 18-24 hoursat 37° C. on L-agar plates. To obtain a seed culture, the strains can begrown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mmtest tubes containing 2 ml of L-broth supplemented with 4% sucrose.Then, the fermentation medium can be inoculated with 0.21 ml of seedmaterial (10%). The fermentation can be performed in 2 ml of a minimalfermentation medium in 20×200-mm test tubes. Cells can be grown for48-72 hours at 32° C. with shaking at 250 rpm. The amount of L-leucinecan be measured by paper chromatography (liquid phase composition:butanol-acetic acid-water=4:1:1).

The composition of the fermentation medium (g/l) (pH 7.2) is as follows:

Glucose 60.0 (NH₄)₂SO₄ 25.0 K₂HPO₄ 2.0 MgSO₄•7H₂O 1.0 Thiamine 0.01CaCO₃ 25.0

Glucose and CaCO₃ are sterilized separately.

Example 6 Production of L-histidine by E. coli 80-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-histidineproduction, DNA fragments from the chromosome of the above-described E.coli MG1655 ΔrcsA::cat can be transferred to the histidine-producing E.coli strain 80 by P1 transduction (Miller, J. H. Experiments inMolecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview,N.Y.) to obtain strain 80-ΔrcsA. The strain 80 has been described inRussian patent 2119536 and deposited in the Russian National Collectionof Industrial Microorganisms (Russia, 117545 Moscow, 1 Dorozhnyproezd, 1) on Oct. 15, 1999 under accession number VKPM B-7270 and thenconverted to a deposit under the Budapest Treaty on Jul. 12, 2004.

Both E. coli strains, 80 and 80-ΔrcsA, can each be cultured in L-brothfor 6 h at 29° C. Then, 0.1 ml of obtained culture can be inoculatedinto 2 ml of fermentation medium in a 20×200-mm test tube and cultivatedfor 65 hours at 29° C. with shaking on a rotary shaker (350 rpm). Aftercultivation, the amount of histidine which accumulates in the medium canbe determined by paper chromatography. The paper can be developed with amobile phase consisting of n-butanol: acetic acid:water=4:1:1 (v/v). Asolution of ninhydrin (0.5%) in acetone can be used as a visualizingreagent.

The composition of the fermentation medium (g/l) is as follows (pH 6.0):

Glucose 100.0 Mameno (soybean hydrolysate) 0.2 of as total nitrogenL-proline 1.0 (NH₄)₂SO₄ 25.0 KH₂PO₄ 2.0 MgSO₄•7H₂0 1.0 FeSO₄•7H₂0 0.01MnSO₄ 0.01 Thiamine 0.001 Betaine 2.0 CaCO₃ 60.0

Glucose, proline, betaine and CaCO₃ are sterilized separately. The pH isadjusted to 6.0 before sterilization.

Example 7 Production of L-glutamate by E. coli VL334thrC⁺-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-glutamateproduction, DNA fragments from the chromosome of the above-described E.coli strain MG1655 ΔrcsA::cat can be transferred to the E. coliL-glutamate-producing strain VL334thrC⁺(EP 1172433) by P1 transduction(Miller, J. H. Experiments in Molecular Genetics, Cold Spring HarborLab. Press, 1972, Plainview, N.Y.) to obtain the strainVL334thrC⁺-ΔrcsA. The strain VL334thrC⁺ has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) (Russia,117545 Moscow, 1 Dorozhny proezd, 1) on Dec. 6, 2004 under the accessionnumber VKPM B-8961 and then converted to a deposit under the BudapestTreaty on Dec. 8, 2004.

Both strains, VL334thrC⁺ and VL334thrC⁺-ΔrcsA, can be grown for 18-24hours at 37° C. on L-agar plates. Then, one loop of the cells can betransferred into test tubes containing 2 ml of fermentation medium. Thefermentation medium contains glucose (60 g/l), ammonium sulfate (25g/l), KH₂PO₄ (2 g/l), MgSO₄ (1 g/l), thiamine (0.1 mg/ml), L-isoleucine(70 μg/ml), and CaCO₃ (25 g/l). The pH is adjusted to 7.2. Glucose andCaCO₃ are sterilized separately. Cultivation can be carried out at 30°C. for 3 days with shaking. After the cultivation, the amount ofL-glutamic acid which is produced can be determined by paperchromatography (liquid phase composition of butanol-aceticacid-water=4:1:1) with subsequent staining by ninhydrin (1% solution inacetone) and further elution of the compounds in 50% ethanol with 0.5%CdCl₂.

Example 8 Production of L-phenylalanine by E. coli AJ12739-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-phenylalanineproduction, DNA fragments from the chromosome of the above-described E.coli MG1655 ΔrcsA::cat can be transferred to the phenylalanine-producingE. coli strain AJ12739 by P1 transduction (Miller, J. H. Experiments inMolecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview,N.Y.) to obtain strain AJ12739-ΔrcsA. The strain AJ12739 has beendeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) onNov. 6, 2001 under accession no. VKPM B-8197 and then converted to adeposit under the Budapest Treaty on Aug. 23, 2002.

Both strains, AJ12739-ΔrcsA and AJ12739, can be cultivated at 37° C. for18 hours in a nutrient broth, and 0.3 ml of the obtained culture caneach be inoculated into 3 ml of a fermentation medium in a 20×200-mmtest tube and cultivated at 37° C. for 48 hours with shaking on a rotaryshaker. After cultivation, the amount of phenylalanine which accumulatesin the medium can be determined by TLC. The 10×15-cm TLC plates coatedwith 0.11-mm layers of Sorbfil silica gel containing no fluorescentindicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used.The Sorbfil plates can be developed with a mobile phase consisting ofpropan-2-ol:ethylacetate:25% aqueous ammonia:water=40:40:7:16 (v/v). Asolution of ninhydrin (2%) in acetone can be used as a visualizingreagent.

The composition of the fermentation medium (g/l) is as follows:

Glucose 40.0 (NH₄)₂SO₄ 16.0 K₂HPO₄ 0.1 MgSO₄•7H₂O 1.0 FeSO₄•7H₂O 0.01MnSO₄ 5H₂O 0.01 Thiamine HCl 0.0002 Yeast extract 2.0 Tyrosine 0.125CaCO₃ 20.0

Glucose and magnesium sulfate are sterilized separately. CaCO₃ isdry-heat sterilized at 180° for 2 hours. The pH is adjusted to 7.0.

Example 9 Production of L-tryptophan by E. coli SV164 (pGH5)-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-tryptophanproduction, DNA fragments from the chromosome of the above-described E.coli strain MG1655 ΔrcsA::cat can be transferred to thetryptophan-producing E. coli strain SV164 (pGH5) by P1 transduction(Miller, J. H. Experiments in Molecular Genetics, Cold Spring HarborLab. Press, 1972, Plainview, N.Y.) to obtain the strain SV164(pGH5)-ΔrcsA. The strain SV164 has the trpE allele encoding anthranilatesynthase free from feedback inhibition by tryptophan. The plasmid pGH5harbors a mutant serA gene encoding phosphoglycerate dehydrogenase freefrom feedback inhibition by serine. The strain SV164 (pGH5) wasdescribed in detail in U.S. Pat. No. 6,180,373 or European patent0662143.

Both strains, SV164 (pGH5)-ΔrcsA and SV164 (pGH5), can each becultivated with shaking at 37° C. for 18 hours in 3 ml of nutrient brothsupplemented with tetracycline (20 mg/l, marker of pGH5 plasmid). Theobtained cultures (0.3 ml each) can be inoculated into 3 ml of afermentation medium containing tetracycline (20 mg/l) in 20×200-mm testtubes, and cultivated at 37° C. for 48 hours with a rotary shaker at 250rpm. After cultivation, the amount of tryptophan which accumulates inthe medium can be determined by TLC as described in Example 8. Thefermentation medium components are listed in Table 2, but should besterilized in separate groups (A, B, C, D, E, F, and H), as shown, toavoid adverse interactions during sterilization.

TABLE 2 Groups Component Final concentration, g/l A KH₂PO₄ 1.5 NaCl 0.5(NH₄)₂SO₄ 1.5 L-Methionine 0.05 L-Phenylalanine 0.1 L-Tyrosine 0.1Mameno (total N) 0.07 B Glucose 40.0 MgSO₄•7H₂O 0.3 C CaCl₂ 0.011 DFeSO₄•7H₂O 0.075 Sodium citrate 1.0 E Na₂MoO₄•2H₂O 0.00015 H₃BO₃ 0.0025CoCl₂•6H₂O 0.00007 CuSO₄•5H₂O 0.00025 MnCl₂•4H₂O 0.0016 ZnSO₄•H₂O 0.0003F Thiamine HCl 0.005 G CaCO₃ 30.0 H Pyridoxine 0.03 Group A had pH 7.1adjusted by NH₄OH. Each group is sterilized separately, chilled, andthen mixed together.

Example 10 Production of L-proline by E. coli 702ilvA-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-prolineproduction, DNA fragments from the chromosome of the above-described E.coli strain MG1655 ΔrcsA::cat can be transferred to theproline-producing E. coli strain 702ilvA by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,1972, Plainview, N.Y.) to obtain the strain 702ilvA-ΔrcsA. The strain702ilvA has been deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhnyproezd, 1) on Jul. 18, 2000 under accession number VKPM B-8012 and thenconverted to a deposit under the Budapest Treaty on May 18, 2001.

Both E. coli strains, 702ilvA and 702ilvA-ΔrcsA, can be grown for 18-24hours at 37° C. on L-agar plates. Then, these strains can be cultivatedunder the same conditions as in Example 8.

Example 11 Production of L-arginine by E. coli 382-ΔrcsA

To test the effect of inactivation of the rcsA gene on L-arginineproduction, DNA fragments from the chromosome of the above-described E.coli strain MG1655 ΔrcsA::cat can be transferred to thearginine-producing E. coli strain 382 by P1 transduction (Miller, J. H.Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972,Plainview, N.Y.) to obtain strain 382-ΔrcsA. The strain 382 has beendeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) onApr. 10, 2000 under accession number VKPM B-7926 and then converted to adeposit under the Budapest Treaty on May 18, 2001.

Both strains, 382-ΔrcsA and 382, can be separately cultivated withshaking at 37° C. for 18 hours in 3 ml of nutrient broth, and 0.3 ml ofthe obtained cultures can be inoculated into 2 ml of a fermentationmedium in 20×200-mm test tubes and cultivated at 32° C. for 48 hours ona rotary shaker.

After the cultivation, the amount of L-arginine which accumulated in themedium can be determined by paper chromatography using the followingmobile phase: butanol:acetic acid:water=4:1:1 (v/v). A solution ofninhydrin (2%) in acetone can be used as a visualizing reagent. A spotcontaining L-arginine can be cut out, L-arginine can be eluted with 0.5%water solution of CdCl₂, and the amount of L-arginine can be estimatedspectrophotometrically at 540 nm.

The composition of the fermentation medium (g/l) is as follows:

Glucose 48.0 (NH4)₂SO₄ 35.0 KH₂PO₄ 2.0 MgSO₄•7H₂O 1.0 Thiamine HCl0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO₃ isdry-heat sterilized at 180° C. for 2 hours. The pH is adjusted to 7.0.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. All the cited referencesherein are incorporated as a part of this application by reference.

Industrial Applicability

According to the present invention, production of an L-amino acid by abacterium of the Enterobacteriaceae family can be enhanced.

The invention claimed is:
 1. A method for producing an L-amino acidcomprising: A) cultivating an L-amino acid-producing Escherichia colibacterium in a medium, and B) collecting said L-amino acid from themedium in an amount of not less than 0.5 g/L, wherein said bacterium hasbeen modified to attenuate expression of the Escherichia coli rcsA genelocated on the chromosome of said bacterium by a method selected fromthe group consisting of: a) deleting a part of the rcsA gene, b)shifting the reading frame of the rcsA gene, c) introducing one or moremissense/nonsense mutations into the rcsA gene, d) modifying sequencescontrolling gene expression of the rcsA gene, and e) combinationsthereof.
 2. The method according to claim 1, wherein said L-amino acidis selected from the group consisting of an aromatic L-amino acid and anon-aromatic L-amino acid.
 3. The method according to claim 2, whereinsaid aromatic L-amino acid is selected from the group consisting ofL-phenylalanine, L-tyrosine, and L-tryptophan.
 4. The method accordingto claim 2, wherein said non-aromatic L-amino acid is selected from thegroup consisting of L-threonine, L-lysine, L-cysteine, L-methionine,L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine,L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid,L-proline, and L-arginine.
 5. The method according to claim 1, whereinsaid rcsA gene encodes a protein having a homology of not less than 95%to the amino acid sequence of SEQ ID NO:
 2. 6. The method according toclaim 5, wherein said L-amino acid is selected from the group consistingof an aromatic L-amino acid and a non-aromatic L-amino acid.
 7. Themethod according to claim 6, wherein said aromatic L-amino acid isselected from the group consisting of L-phenylalanine, L-tyrosine, andL-tryptophan.
 8. The method according to claim 6, wherein saidnon-aromatic L-amino acid is selected from the group consisting ofL-threonine, L-lysine, L-cysteine, L-methionine, L-leucine,L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline,and L-arginine.
 9. A method for producing an L-amino acid comprising: A)cultivating an L-amino acid-producing Escherichia coli bacterium in amedium, and B) collecting said L-amino acid from the medium in an amountof not less than 0.5 g/L, wherein said bacterium has been modified toattenuate expression of the Escherichia coli rcsA gene located on thechromosome of said bacterium by a method selected from the groupconsisting of: a) deleting a part of or the entire rcsA gene, b)shifting the reading frame of the rcsA gene, c) introducing one or moremissense/nonsense mutations into the rcsA gene, d) modifying sequencescontrolling gene expression of the rcsA gene, and e) combinationsthereof.